The yeast ATF/CREB repressor Sko1(Acr1) regulates genes that are induced upon hyperosmotic stress by recruiting the Cyc8(Ssn6)-Tup1 corepressor complex to target promoters. During hyperosmotic stress, Hog1 MAP kinase associates with target promoters, phosphorylates Sko1, and converts Sko1 into a transcriptional activator. Unexpectedly, Tup1 remains bound to target promoters during osmotic stress. Sko1, Hog1, and Tup1 are all important for recruitment of SAGA histone acetylase and SWI/SNF nucleosome-remodeling complexes to osmotic-inducible promoters, and both complexes are important for activation upon osmotic stress. Thus, osmotic induction involves a switch of Sko1-Cyc8-Tup1 from a repressing to an activating state in a process that is triggered by Hog1 phosphorylation. Cyc8-Tup1 is not simply a corepressor but is also involved in recruiting SWI/SNF and SAGA during the transcriptional induction process.

The yeast ENA1/PMR2A gene encodes a cation extrusion ATPase in Saccharomyces cerevisiae which is essential for survival under salt stress conditions. One important mechanism of ENA1 transcriptional regulation is based on repression under normal growth conditions, which is relieved by either osmotic induction or glucose starvation. Analysis of the ENA1 promoter revealed a Mig1p-binding motif (؊533 to ؊544) which was characterized as an upstream repressing sequence (URS MIG-ENA1 ) regulated by carbon source. Its function was abolished in a mig1 mig2 double-deletion strain as well as in either ssn6 or tup1 single mutants. A second URS at ؊502 to ؊513 is responsible for transcriptional repression regulated by osmotic stress and is similar to mammalian cyclic AMP response elements (CREs) that are recognized by CREB proteins. This URS CRE-ENA1 element requires for its repression function the yeast CREB homolog Sko1p (Acr1p) as well as the integrity of the Ssn6p-Tup1p corepressor complex. When targeted to the GAL1 promoter by fusing with the Gal4p DNA-binding domain, Sko1p acts as an Ssn6/Tup1p-dependent repressor regulated by osmotic stress. A glutathione S-transferase-Sko1 fusion protein binds specifically to the URS CRE-ENA1 element. Furthermore, a hog1 mitogen-activated protein kinase deletion strain could not counteract repression on URS CRE-ENA1 during osmotic shock. The loss of SKO1 completely restored ENA1 expression in a hog1 mutant and partially suppressed the osmotic stress sensitivity, qualifying Sko1p as a downstream effector of the HOG pathway. Our results indicate that different signalling pathways (HOG osmotic pathway and glucose repression pathway) use distinct promoter elements of ENA1 (URS CRE-ENA1 and URS MIG-ENA1 ) via specific transcriptional repressors (Sko1p and Mig1/2p) and via the general Ssn6p-Tup1p complex. The physiological importance of the relief from repression during salt stress was also demonstrated by the increased tolerance of sko1 or ssn6 mutants to Na ؉ or Li ؉ stress.

Exposure of yeast to increases in extracellular osmolarity activates the Hog1 mitogen‐activated protein kinase (MAPK), which is essential for the induction of gene expression required for cell survival upon osmotic stress. Several genes are regulated in response to osmotic stress by Sko1, a transcriptional repressor of the ATF/CREB family. We show by in vivo coprecipitation and phosphorylation studies that Sko1 and Hog1 interact and that Sko1 is phosphorylated upon osmotic stress in a Hog1‐dependent manner. Hog1 phosphorylates Sko1 in vitro at multiple sites within the N‐terminal region. Phosphorylation of Sko1 disrupts the Sko1–Ssn6–Tup1 repressor complex, and consistently, a mutant allele of Sko1, unphosphorylatable by Hog1, exhibits less derepression than the wild type. Interestingly, Sko1 repressor activity is further enhanced in strains with high protein kinase A (PKA) activity. PKA phosphorylates Sko1 near the bZIP domain and mutation of these sites eliminates modulation of Sko1 responses to high PKA activity. Thus, Sko1 transcriptional repression is controlled directly by the Hog1 MAPK in response to stress, and this effect is further modulated by an independent signaling mechanism through the PKA pathway.

The expression of gluconeogenic fructose-1,6-bisphosphatase (encoded by the FBP1 gene) depends on the carbon source. Analysis of the FBP1 promoter revealed two upstream activating elements, UAS1FBP1 and UAS2FBP1, which confer carbon source-dependent regulation on a heterologous reporter gene. On glucose media neither element was activated, whereas after transfer to ethanol a 100-fold derepression was observed. This gene activation depended on the previously identified derepression genes CAT1 (SNF1) (encoding a protein kinase) and CAT3 (SNF4) (probably encoding a subunit of Cat1p [Snf1p]). Screening for mutations specifically involved in UAS1FBP1 derepression revealed the new recessive derepression mutation cat8. The cat8 mutants also failed to derepress UAS2FBP1, and these mutants were unable to grow on nonfermentable carbon sources. The CAT8 gene encodes a zinc cluster protein related to Saccharomyces cerevisiae Gal4p. Deletion of CAT8 caused a defect in glucose derepression which affected all key gluconeogenic enzymes. Derepression of glucose-repressible invertase and maltase was still normally regulated. A CAT8-lacZ promoter fusion revealed that the CAT8 gene itself is repressed by Cat4p (Mig1p). These results suggest that gluconeogenic genes are derepressed upon binding of Cat8p, whose synthesis depends on the release of Cat4p (Mig1p) from the CAT8 promoter. However, gluconeogenic promoters are still glucose repressed in cat4 mutants, which indicates that in addition to its transcription, the Cat8p protein needs further activation. The observation that multicopy expression of CAT8 reverses the inability of cat1 and cat3 mutants to grow on ethanol indicates that Cat8p might be the substrate of the Cat1p/Cat3p protein kinase.

Regulation of gene expression by stress-activated protein kinases (SAPKs) is essential for cell adaptation to extracellular stimuli. Exposure of yeast to high osmolarity results in activation of the SAPK Hog1, which associates with transcription factors bound at target promoters and stimulates transcriptional initiation. Unexpectedly, activated Hog1 also associates with elongating Pol II and components of the elongation complex. Hog1 is selectively recruited to the entire coding region of osmotic stress genes, but not to constitutively expressed genes. Selective association of Hog1 with the transcribed region of osmoresponsive genes is determined by the 3' untranslated region (3' UTR). Lastly, Hog1 is important for the amount of the RNA polymerase II (Pol II) elongation complex and of mRNA produced from genes containing osmoresponsive coding regions. Thus, in addition to its various functions during transcriptional initiation, Hog1 behaves as a transcriptional elongation factor that is selective for genes induced upon osmotic stress.

In yeast, hyperosmotic stress causes an immediate dissociation of most proteins from chromatin, presumably because cells are unprepared for, and initially unresponsive to, increased ion concentrations in the nucleus. Osmotic stress activates Hog1 MAP kinase, which phosphorylates at least two proteins located at the plasma membrane, the Nha1 Na+/H+ antiporter and the Tok1 potassium channel. Hog1 phosphorylation stimulates Nha1 activity, and this is crucial for the rapid reassociation of proteins with their target sites in chromatin. This initial response to hyperosmolarity precedes and temporally regulates the activation of stress-response genes that depends on Hog1 phosphorylation of transcription factors in the nucleus. Thus, a single MAP kinase coordinates temporally, spatially, and mechanistically distinct responses to stress, thereby providing very rapid stress relief that facilitates subsequent changes in gene expression that permit long-term adaptation to harsh environmental conditions.

A major part of the transcriptional response of yeast cells to osmotic shock is controlled by the HOG pathway and several downstream transcription factors. Sko1p is a repressor that mediates HOG pathway-dependent regulation by binding to CRE sites in target promoters. Here, we report five target genes of Hog1p-Sko1p: GRE2, AHP1, SFA1, GLR1 and YML131w. The two CREs in the GRE2 promoter function as activating sequences and, hence, bind (an) activator protein(s). However, the two other yeast CRE-binding proteins, Aca1p and Aca2p, are not involved in regulation of the GRE2 promoter under osmotic stress. In the absence of the co-repressor complex Tup1p-Ssn6p/Cyc8p, which is recruited by Sko1p, stimulation by osmotic stress is still observed. These data indicate that Sko1p is not only required for repression, but also involved in induction upon osmotic shock. All five Sko1p targets encode oxidoreductases with demonstrated or predicted roles in repair of oxidative damage. Altered basal expression levels of these genes in hog1Delta and sko1Delta mutants may explain the oxidative stress phenotypes of these mutants. All five Sko1p target genes are induced by oxidative stress, and induction involves Yap1p. Although Sko1p and Yap1p appear to mediate osmotic and oxidative stress responses independently, Sko1p may affect Yap1p promoter access or activity. The five Sko1p target genes described here are suitable models for studying the interplay between osmotic and oxidative responses at the molecular and physiological levels.

The Cat8p zinc cluster protein is essential for growth of Saccharomyces cerevisiae with nonfermentable carbon sources. Expression of the CAT8 gene is subject to glucose repression mainly caused by Mig1p. Unexpectedly, the deletion of the Mig1p-binding motif within the CAT8 promoter did not increase CAT8 transcription; moreover, it resulted in a loss of CAT8 promoter activation. Insertion experiments with a promoter test plasmid confirmed that this regulatory 20-bp element influences glucose repression and derepression as well. This finding suggests an upstream activating function of this promoter region, which is Mig1p independent, as ⌬mig1 mutants are still able to derepress the CAT8 promoter. No other putative binding sites such as a Hap2/3/4/5p site and an Abf1p consensus site were functional with respect to glucose-regulated CAT8 expression. Fusions of Cat8p with the Gal4p DNA-binding domain mediated transcriptional activation. This activation capacity was still carbon source regulated and depended on the Cat1p (Snf1p) protein kinase, which indicated that Cat8p needs posttranslational modification to reveal its gene-activating function. Indeed, Western blot analysis on sodium dodecyl sulfate-gels revealed a single band (Cat8pI) with crude extracts from glucose-grown cells, whereas three bands (Cat8pI, -II, and -III) were identified in derepressed cells. Derepression-specific Cat8pII and -III resulted from differential phosphorylation, as shown by phosphatase treatment. Only the most extensively phosphorylated modification (Cat8pIII) depended on the Cat1p (Snf1p) kinase, indicating that another protein kinase is responsible for modification form Cat8pII. The occurrence of Cat8pIII was strongly correlated with the derepression of gluconeogenic enzymes (phosphoenolpyruvate carboxykinase and fructose-1,6-bisphosphatase) and gluconeogenic PCK1 mRNA. Furthermore, glucose triggered the dephosphorylation of Cat8pIII, but this did not depend on the Glc7p (Cid1p) phosphatase previously described as being involved in invertase repression. These results confirm our current model that glucose derepression of gluconeogenic genes needs Cat8p phosphorylation and additionally show that a still unknown transcriptional activator is also involved.